The long term goal of this proposal is to understand how the structure and energetics of the DNA template contribute to regulatory mechanisms for the initiation of DNA replication and transcription in living cells. Supercoiling of the DNA template is energetically unfavorable and can result in localized opening or unwinding of the helix in vitro. This proposal is a continuation of our characterization of the single-strand- specific endonucleases, mung bean nuclease and P1 nuclease, as probes for unwinding in naturally-occurring DNA sequences present in supercoiled plasmids and viral genomes. The enzymes recognize Z-DNA, cruciforms and a novel, stably-unwound DNA conformation call the AT-rich structure. The AT- rich structure occurs in several prokaryotic and eukaryotic replication origins. Extensive mutational analysis of a yeast replication origin revealed that detection of the AT-rich structure in vitro correlates with the ability to initiate DNA replication in vivo. The specific aims of this proposal are to 1) examine the architecture, DNA sequence selectivity and energetics of the AT-rich structure, 2) investigate easily-unwound DNA sequences as determinants for initiation of replication in vivo, and 3) probe for localized DNA unwinding in chromatin. The hypothesis that the AT-rich structure is not melted but rather a lower energy DNA conformation which is partially untwisted and base paired, will be tested by 1) two dimensional gel electrophoresis of topoisomers to assess the energy and extent of DNA unwinding, and 2) analysis of DNA sequences which react with the enzymes as well as chemical probes for paired or unpaired bases. They hypothesis that the low energy of DNA unwinding is a determinant of replication initiation in vivo will be tested by comparing the effects of mutations on formation of an AT-rich structure in the chromosomal origin (oriC) of E. coil and the simian virus 40 (SV40) origin in vitro with the ability of the mutant derivatives to initiate replication in vivo. Finally, we will establish the use of mung bean nuclease and P1 nuclease as chromatin probes for unpaired and non-Watson-Crick paired bases, in SV40 chromatin at nucleotide level resolution.